nutritional properties of microalgae for mariculture

8/11/2019 Nutritional Properties of Microalgae for Mariculture

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Table 1Microalgae commonly used as larval feeds for specific animals in Australian mariculture

Maricultured animal species Commonly used algae

Oyster larvae: Crassostrea gigas,

Saccostrea commerci al i s,

Pinctada maxima

Scallop larvae: Pecten spp.Clam larvae: Tri dacna gi gasAbalone juvenile: Hal io t i s spp.Prawn larvae: Penaeus spp.

Zooplankton b: Brachionis plicati l i s,Anemia spp.

Isochtysis sp. (TISO), Pauloua lut heri , Thalassiosira pseudonana,Chaet oceros muell eri a, Chaet oceros calci t runs

Isochrysis sp. (TJSO), Chaetoceros muelleri, Chaetoceros calcitransIsochrysis sp. (TISO), Paulova l utheri, Chaetoceros muel leriNauicula sp. (CS-461, Amphora sp. (CS-lo), Ni tzschia closteriumSkel et onema costat um, Sk el et onema sp. (CS-2521, Tet rasel mi s chui,Tet rasel mi s suecica, Chaet oceros muell eri

lsochtysis sp. (T.ISO), Pavl ova lut heri , Nannochlor opsi s oculat a

a This strain was originally provided to CSIRO as Chaetoceros gracilis Schitt (designation CHGRA) from theProvasoli-Guillard National Center for Culture of Marine Phytoplankton and is widely known as such by theAustralian mariculture industry. It has subsequently been correctly identified as Chaetoceros muelleriLemmermann 1898.b Used as food for fish, prawn larvae.

pm). Exceptions are diatom chains (e.g. Skeletonemu spp.: up to 60 km long) forraptorial feeders such a prawn larvae, and sticky pennate diatoms (e.g. Nitzschia spp.and Nuvicula spp.: > 20 p,m> for grazers such as abalone. Microalgae that originallywere isolated from northern hemisphere waters are used in most of Australias 50

hatcheries; their success as food species depends on their nutritional quality, as well astheir tolerance to temperature, salinity and light, especially if grown in outdoor tanks orponds. Not all overseas species have been successful in the Australian environment.Improved microalgal production for mariculture in Australia requires- knowledge of the optimal growth responses of microalgae under local conditions;- knowledge of the nutritional value of microalgae matched to the nutritional require-

ments of animals; and* isolation of new microalgal strains more suited to local conditions.

In 1986 we started a programme (funded by the Fisheries Industry and DevelopmentCorporation) to examine microalgal feedstocks used in Australian mariculture (Jeffrey

and Garland, 1987). To date, the gross composition, amino acids, lipid classes, fattyacids, sugars and vitamins of about 40 species from seven algal classes have beenexamined to assess how composition relates to differences in the nutritional value of thespecies. In the present paper, we review these studies. Our studies on the environmentaltolerances of the microalgae are presented elsewhere (Jeffrey et al., 1992)

2. Methods

2 1 Algal culture

The biochemical composition of over 40 species of microalgae from the CSIROAlgal Culture Collection that are useful (or potentially useful) in Australian mariculturewere analysed. Representatives of most of the major algal taxa were examined: diatoms,


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M R. Brow n et al . Aquacul t ure 151 1997) 315-331 317

prymnesiophytes, prasinophytes, chiorophytes, eustigmatophytes, cryptomonads and arhodophyte (Table 2). Experimental scale cultures were used for our baseline studies.Cultures were grown in 1.2 1 of culture medium in 2 1 Erlenmeyer flasks on glass

shelves illuminated from beneath with 70-80 PE mm2 s-r white fluorescent light on a12: 12 h 1ight:dark cycle. Temperate species were maintained at 20C and tropicalspecies were grown at 25C. In initial studies, cultures were not aerated, but were gentlyswirled on an orbital shaker (Volkman et al., 1989, 1993; Brown, 1991; Brown andJeffrey, 1992a; Dunstan et al., 1992). In subsequent studies, cultures were gently aeratedwith 1% CO, in air (Brown and Miller, 1992; Brown and Farmer, 1994; Brown et al.,unpublished results).

The cultures were harvested towards the end of log phase (usually 4-6 h into thelight cycle), and in some later studies (Brown and Miller, 1992; Brown and Farmer,1994) stationary phase cultures also were examined. Cells were counted (six replicatecounts per species) with a Neubauer haemocytometer. The length, width or diameter ofat least five representative living cells in each culture were measured. Volumes werecalculated according to the basic geometry of the cell (e.g. sphere, oblate ellipsoid andrectangular box) from equations given by Smayda (1978).

Aliquots were collected for the analysis of dry weight (2 X 100 ml), chlorophyll a(2 X 5 ml), fatty acids (250 ml) and (when analysed) vitamins (2 X 100 ml). Theremainder of the culture was harvested by centrifugation (5000 X g for 10 min). Thecells were washed with 100 ml of 0.5 M ammonium formate (to remove residual saltsfrom the seawater medium) and recentrifuged. The supematant was discarded. The cellpellet was freeze-dried, and then stored at - 20C for other chemical analyses.

Three studies determined the effects of culture conditions on the biochemicalcomposition: stage of harvest (Brown et al., 1993a; Dunstan et al., 1993) light intensity(Brown et al., 1993b) and light intensity and photoperiod (Brown et al., unpublishedresults). Full details of the growth conditions of individual species and harvestingprotocols are given in the relevant publications.

2.2. Procedures for biochemical analyses

Sensitive and specific analytical methods were required to measure the nutrientconcentrations in the small quantities of microalgal samples available (I 20 mg dryweight).

For amino acids, algal samples were hydrolysed at 110C for 24 h with 4 Mmethanesulphonic acid, in hydrolysis tubes. Hydrolysates were then passed through acation-exchange resin (AG SOW-X8) and purified amino acids eluted with 2 Mammonium hydroxide (Lazarus, 1973). The amino acids were then reacted with phenylisothiocyanate (Bidlingmeyer et al., 1984) and analysed by reverse-phase high-perfor-mance liquid chromatography (HPLC) (Yang and Sepulveda, 1985). The anhydroaminoacid residues were summed to give an estimate of the protein content.

For lipid, algae were extracted with a chloroform-methanol-water mixture (2:4:1)

(Whyte, 1987). The resultant filtrates were combined, and separated into chloroform andaqueous-methanol layers by the addition of water and chloroform. The chloroform layerswere then concentrated and weighed to determine the total lipid. Fatty acids in the lipid


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+++++++++++i+++++

+++++++++++c++*+++ / I I I I

+ +++++ ++ +++++++++


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extract were transesterified to methyl esters and analysed by capillary gas chromatogra-phy (GC) using polar and non-polar capillary columns and GC-mass spectrometry(Volkman et al., 1989). Lipid classes were determined by thin-layer chromatography

with flame ionization detection (Iatroscan TLC-FID) (Volkman and Nichols, 1991).The aqueous-methanol layers (from the above lipid extractions) contained mono- and

oligosaccharides, and were assayed for carbohydrate by the phenol-sulphuric acidmethod (Dubois et al., 1956). The residues remaining after lipid extraction containedpolysaccharide, and were hydrolysed with 0.5 M H,SO, at 100C. Portions wereassayed for carbohydrate (Dubois et al., 1956). Total carbohydrate was determined bysumming the mono-, oligosaccharide carbohydrate and polysaccharide carbohydrate.

The remaining portions of the hydrolysates from the polysaccharide fractions wereneutralized and desalted (Brown, 1991). Eluants were freeze-dried, the sugars wereconverted to alditol-acetate derivatives and analysed by GC (Blakeney et al., 1983).

The vitamins, ascorbic acid and riboflavin, were assayed by reverse-phase HPLCwith fluorimetric detection (Brown and Miller, 1992; Brown and Farmer, 1994).

3. Results and discussion

The nutritional properties of the microalgae, grown under standard conditions, aresummarized in Table 2. More detailed information is given in the references cited in thetable. Key findings for individual species, and trends across taxonomic classes, are givenbelow.

3 I Gross composition

Classes of microalgae differed appeciably in their content of protein, carbohydrateand lipid (Fig. 1). In our initial screening studies with non-aerated (static) cultures,protein was the major organic component (6-34% of total dry weight), followed by lipid(7-23%) and carbohydrate (5-23%) (Fig. l(A)). There were no class-specific differ-ences in protein and lipid, but the chlorophytes and prasinophytes were typically richerin carbohydrate than species from other classes.

In later studies, we cultured microalgae under aeration with 1% CO, in air, tosimulate the conditions required for the culture of large volumes (e.g. 500 1) in hatcheryoperations. The gross composition of these cultures had, on average, similar levels oflipid and carbohydrate to static cultures (Fig. l(B)). However, the average protein level(34%) was greater than for static cultures (24%). Protein was in the range 15-52%, lipid5-20% and carbohydrate 5-12%. Diatoms contained more lipid than other algae, onaverage.

The gross composition of many of the microalgal classes was unrelated to nutritionalvalues reported previously. Phaeodactylum tricornutum and Nannochloris atomus wererich in protein and carbohydrate, respectively, but these species generally are regarded

as having low food value (Brown et al., 1989). Gross composition may not alwayscorrelate directly with nutritional value (Webb and Chu, 1983; Brown et al., 1989), butwhen other specific essential nutrients (e.g. polyunsaturated fatty acids, vitamins) are in


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M.R. Brown et al./Aquaculture 151 1997) 315-331 321

A) Non-aerated cultures

Diatoms (6)

Prymnesiophytes (4)

Eustigmatophytes (4)

Cryptomonads (1)

Prasinophytes (7)

Chlorophytes (5)

Rhodophyte (1)

All species

0 10 20 30 40 50

protein

5 10 15 20 0 5 IO 15 20 25

lipid carbohydrate

B) Cultures aerated with 1 COP

Diatoms (7)

Prymnesiophytes (2)


Cryptomonads (7)

All speciesII 1 I I I I

0 10 20 30 40 50

protein

t I I I I0 5 10 15 20

lipid

Fig. 1. Percentages (dry weight basis) of protein, lipid and carbohydrate in microalgae: A, non-aerated

A ; lb 1; ;ocarbohydrate

/5

cultures; B, cultures aerated with 1% CO,. The range of values is shown by range bars.

adequate proportion, the differences may become important. Algal diets rich in poly-unsaturated fatty acids and with high levels of carbohydrate are reported to produce thebest growth for juvenile oysters Ostrea edulis; Enright et al., 1986) and larval scallopsPutinopecten yessoensis; Whyte et al., 1989), while high dietary protein provides best

growth for juvenile mussels Myths trossulus; Kreeger and Langdon, 1993).

Although the differences in composition of many of the species might reflect geneticdifferences, these might sometimes be attributed to the culture conditions. Lightintensities and photoperiod were identical for all cultures, but several different culture


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M.R. Bra,

Eustigmatophytes

Diatoms

Prymnesiophytes

CryptomonadsRhodophyte

Chlorophytes

Prasinophytes

All species

Oyster larvae

et al./Aquaculture 151 1997) 315-331 323

0 5 10 150 1 2 30 2 4 60 5 IO

arginine

Eusttgmatophytes

Diatoms

Prymnesiophytes

Cryptomonads

Rhodophyte

Chlorophytes

Prasinophytes

All species

Oyster larvae

0 3 6lysine

Eustigmatophytes

Diatoms

Prymnesiophytes

Cryptomonads

Rhodophyte

Chlorophytes

Prasinophytes

All species

Oyster larvae

0 2 4 6threonine

hi&dine isoleucine

0 1 2 3methionine

0 1 2 3tryptophan

0 2 4 6 8

phenylalanine

0 2 4 6valine

leucine

0 5 10 1proline

Fig. 2. Essential amino acid composition (weight percentage of total amino acids) of microalgae compared tooyster Crassostrea gigas) larvae. The range of values is shown by range bars.

classes, Prymnesiophytes, however, contained more arabinose (2-12%), on average,than microalgae from other classes (O-2%).

The efficiency with which marine animals digest polysaccharide is dependent on thepolysaccharide type: i.e. the sugar composition and how the sugars are linked to eachother (Kristensen, 1972; Onishi et al., 1985). Although the digestibility of algalpolysaccharide is not known, the glucose-rich polysaccharide found in most microalgae

should be effectively digested by amylase present in the digestive organs of molluscsand crustaceans (Kristensen, 1972). However, we found Phaeodactylum tricomutum tobe unique in its high concentration of mannose (46%). This alga is reported to have a


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324 M.R. Brown et al. /Aquaculture 151 1997) 315-331

Diatoms (7)

Prymnesiophytes (4)


Cryptomonads (1)

Prasinophytes (7)

Chlorophytes (5)

0 20 40 60 60

glucose

Diatoms (7)

Prymnesiophytes (4)


Cryptomonads (1)

Prasinophytes (7)

Chlorophytes (5)

0 4 6 12 16

fucose

0 10203040

mannose E4 8 121620 0 4 6 12 18galactose0246810 0 2 4 8 8

xylose ribose

rhamnose

A

0 3 6 9 12

arabinose

Fig. 3. The sugar composition (weight percentage of total sugars) of polysaccharide from the microalgaeexamined. Histograms represent average values from the algal class; range of values is shown by range bars.

low nutritional value for bivalve molluscs (Enright et al., 1986); possibly the mannose-rich polysaccharide in the algae makes it less digestible for these animals.

Studies by other investigators have shown that the sugar composition of microalgaecan differ between logarithmic and stationary phase cultures, but the differences arespecies-specific (Chu et al., 1982; Whyte, 1987).

3.4. Vitamins

Levels of ascorbic acid (vitamin C) in 11 microalgal species, across logarithmic andstationary growth phases, ranged from 1.1 (Thalassiosiru pseudonana) to 16 mg g - dryweight (Chaetoceros muelleri) (Fig. 4; Brown and Miller, 1992). Levels were unrelatedto algal class. Many of the species had different levels of ascorbic acid betweenlogarithmic and stationary phases. Chaetoceros muelleri, Thalassiosira pseudonana,Nunnochloropsis oculatu and Zsochrysis sp. (T-ISO) had more ascorbic acid during thelogarithmic phase, whereas Dunaliella tertiolecta and Nannochloris atomus had moreascorbic acid during the stationary phase. Despite the 15fold range in the ascorbic acidcontent of microalgae, all the species should provide an adequate supply of ascorbic acidto cultured animals, which are reported to require only 0.03-0.2 mg g- of the vitaminin their diet (Durve and Lovell, 1982; Shigueno and Itoh, 1988).

Transfer of ascorbic acid (and other vitamins) between trophic levels is important forfish larvae and late larval/early juvenile crustaceans that are reared on algal-fedzooplankton. Hapette and Poulet (1990) fed microalgae to previously starved popula-


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MR. Brown et al./Aquaculture 151 1997) 315-331 325

Chaetoceros calcitrans

Chaetoceros muelleri

Skeletonema costatum

Thalassiosira pseudonana

Dunaliella tertiolecta

Nannochloris atomus

Rhodomonas salina

Nannochloropsis oculata

Tetraselmis suecica

lsochrysis sp. T.lSO)

Pavlova lutheri

5 10 15 20

Proportion of ascorbic acid (mg/g dry weight)

Fig. 4. Content of ascorbic acid in the microalgae.

tions of the copepods Calanus helgolandicus and Acartia clausi, and noted increases inthe ascorbic acid levels of 50% and 60%, respectively, for the animals. Their resultssupported the hypotheses that ascorbic acid from the microalgae can be incorporatedwith high efficiency through direct feeding. All the 11 species we examined, if ingested

and digested, should provide an adequate supply of ascorbic acid to cultured animals atthe next trophic level (e.g. fish larvae) of the mariculture food chain.

Microalgae also were rich in riboflavin. In a study of six species, we foundconcentrations during logarithmic growth-phase ranged from 20 p,g g- dry weightThalassiosira pseudonana) to 40 pg g- Zsochrysis sp. (T.ISO)) (Fig. 5; Brown and

Farmer, 1994). With the onset of stationary phase, the proportion of riboflavin increased

Chaetoceros muelleri

logariihmic phase;

Thalassiosira pseudonana

Nannochloris atomus

Nannochloropsis oculata

lsochtysis sp. T.ISO)

Pavlova lutheri

0 20 40 60 80 100 120

Proportion of riboflavin @g g-l dry weight)

Fig. 5. Content of riboflavin in the microalgae.


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differences according to taxonomic group, although there were examples of differencesbetween species from the same algal class.

Diatoms, eustigmatophytes, cryptomonads, rhodophytes and some prymnesiophytes

(Puuloua spp.) were all rich sources of 20:5(n - 3) (7-34%) (Fig. 6; Volkman et al.,1989, 1991, 1993; Dunstan et al., 1992, 1994; unpublished data). Many representativesfrom these classes have been used successfully as food in larval culture (reviewed inBrown et al., 1989). Cryptomonads and prymnesiophytes also were relatively rich in22:6(n - 3) (0.2-ll%), whereas eustigmatophytes, rhodophytes and diatoms werehighest in 20:4(n - 6) (O-9%). Chlorophytes were deficient in both C,, and C,,PUFAs, although some species had small amounts of 20:5(n - 3) (up to 3.2%).Chlorophytes generally have low nutritional value and are not suitable as a singlespecies diet (Brown et al., 1989), probably because of their deficiencies in the C,, andC,, PUFAs. Prasinophyte species contained significant proportions of C,, and C,,PUFAs (but rarely both). Prasinophyte species such as Tetruselmis spp. have been usedsuccessfully for prawn and mollusc culture (reviewed by Brown and Jeffrey, 1992b).

The PUFAs 18:2(n - 6) and/or 18:3(n - 3) are essential for many freshwater fish(Caste11 et al., 1986). The dietary C,, PUFA can be elongated and desaturated to formlonger chain PUFA more efficiently by freshwater fish than by marine fish, althoughdietary 20:5(n - 3) and 22:6(n - 3) when available, are preferentially incorporated intofish tissues. Significant levels of 18:2(n - 6) and 18:3(n - 3) were found in mostmicroalgal groups, except the diatoms and eustigmatophytes which contained very lowlevels (Volkman et al., 1989, 1993; Dunstan et al., 1994). The major C,, PUFA inprasinophytes and prymnesiophytes was 18:4(n - 3) (Volkman et al., 1989; Dunstan etal., 1992). The significance of these polyunsaturated C ,s PUFA in marine finfish dietsare unknown.

Thompson et al. (1993) found a correlation between the percentage composition ofthe short chain fatty acids 14:0 + 16:0 in microalgae, and the growth rates of Pacificoyster Crussostreu gigus) larvae fed on the microalgae. They reasoned that diets withhigher levels of the saturated fats were more nutritious for the rapidly growing larvae,because energy is released more efficiently from saturated fats than unsaturated fats.Prymnesiophytes, on average, contain the highest proportions of saturated fats (33%),followed by diatoms and eustigmatophytes (27%), chlorophytes and prasinophytes

(23%) and cryptomonads (18%) (Volkman et al., 1989, 1991, 1993; Dunstan et al.,1992, 1994; unpublished data).Fatty acids from microalgae may be efficiently transferred to higher trophic levels

(e.g. to fish larvae) via intermediary zooplankton (Watanabe et al., 1983). For marinefish larvae, the most popular microalgae for boosting zooplankton intermediates withPUFAs are those which contain high levels of 20:5(n - 3) (e.g. Nunnochloropsisoculu t u), 22:6 n - 3) e.g. Z sochr ysi s sp. clone T-ISO) or both (e.g. PuvZovu lutheri).Rotifers (Bruchionus pl icut i l i s ) readily ingest PuuZouu cells from which they canaccumulate high concentrations of 20:5(n - 31, 22:6(n - 3) and other PUFAs within afew hours (Nichols et al., 1989). After just 3 h feeding on Puulouu, the distribution of

fatty acids in the rotifer becomes almost indistinguishable from its algal food.The fatty acid composition of microalgae is dependent on the growth conditions and

the stage of harvest. Logarithmic phase cultures of the diatom Thulussiosiru pseudonunu


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328 MR. Brown et al./Aquaculture I.71 1997) 318-331

grown under a 12:12 h 1ight:dark regime (100 p,E mm2 s- > contained a 25% greaterproportion of 20:5(n - 3) than similar cultures grown under continuous light (50 and100 FE me2 so) (Brown et al., 1996). Nannochloropsis oculatu contained more

PUFA per cell in logarithmic phase than in stationary phase, whereas the reverse wastrue for Puulo~~a lutheri (Dunstan et al., 1993). Other studies have demonstrated changesin fatty acid composition associated with light intensity (Thompson et al., 1990, 1993;Brown et al., 1993b), culture media (Ben-Amotz et al., 1985), temperature (Thompson etal., 1992) and pH (Guckert and Cooksey, 1990), but many of the changes werespecies-specific.

The lipid class composition of microalgae also can vary with culture condition andgrowth phase. Lipids of microalgae are rich in polar lipids during logarithmic phase, butaccumulate triacylglycerol during stationary phase (Dunstan et al., 1993).

3.6. Nutritional evaluation of new Australian microalgae

As our studies included Australian isolates from the major algal classes, we cancompare their profiles with similar strains from overseas that are currently being used inAustralian mariculture. Full data for these species are given in our publications, but datafor the 15 most promising species are summarised below.

Diatoms. A tropical Skeletonema sp. (CS-252) had a similar profile of grosscomposition, amino acids and fatty acids to the overseas temperate strain Skeletonemucostatum (CS-18 1). Two benthic diatoms, Nitzschia closterium CS-SC) and NuvicuZusp. (CS-46c), were rich in protein and good sources of 20:5(n - 3) (Dunstan et al.,1994; Brown and Jeffrey, unpublished results).

Eustigmatophytes. A tropical Nannochloropsis-like species (CS-246) had high con-centrations of the essential fatty acid 20:5 n - 3) (Volkman et al., 1993). It had a similarcomposition to Nunnochloropsis oculatu (CS-1791, an overseas strain used widely inrotifer culture.

Prasinophytes. The tropical Micromonas pusilla CS-170) had low concentrations ofprotein. However, it was a good source of 22:6 n - 3), as was Pyramimonas cordataCS-140). An unidentified coccoid prasinophyte (CS-126) contained significant amounts

of 20:5 n - 3) (Brown and Jeffrey, 1992a; Dunstan et al., 1992).Chlorophytes. Two Chlorella spp. (CS-247 and CS-195) and Stichococcus sp.

(CS-92) lacked the long-chain PUFAs similar to other chlorophyte species. However,they were rich in the C,, and C,, PUFAs, and may be useful in a mixed algal diet(Brown and Jeffrey, 1992a; Dunstan et al., 1992).

Cryptomonads. Rhodomonas salina CS-24), Rhodomonas maculata CS-851, anunidentified cryptomonad from Queensland (CRFlOl) and a temperate cryptomonad(CRPLOl) isolated from Pipeclay Lagoon, Tasmania all were rich in protein (Brown etal., unpublished results) and PUFAs (Dunstan et al., unpublished results), and weresimilar in composition to the overseas strain Rhodomonas salina (CS-174).

Prymnesiophytes. A Pavlova sp. (CS-63) isolated from Port Phillip Bay, which

grows well at 25-30C had high concentrations of the essential PUFAs 20:5 n - 3) and22:6 n - 3); it resembled the commonly used overseas strain Puulova lutheri CS-182)(Volkman et al., 1991).


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4 Concluding remarks

329

Microalgae vary appreciably in their biochemical composition, even when grown

under standard conditions. Gross composition differs between species, but for manyspecies this is not the major factor relating to food value. The protein quality of allmicroalgae is high. Sugar composition is variable, and in some instances may affect thenutritional value. The essential PUFAs 20:5( n - 3) and 22:6( y1 3) are key nutrients inanimal nutrition, and most algae are rich in one or both of these acids. Chlorophytes,however, lack these acids and this contributes to their low food value. Microalgae arerich sources of two key vitamins, ascorbic acid and riboflavin, but some species lackspecific vitamins (De Roeck-Holtzhauer et al., 1991). Because microalgae may belimiting in one or more of the key nutrients, mixed-algal diets provide a better balanceand normally are used in mariculture. The nutritional profiles of Australian isolates

matched those of similar overseas strains closely.The biochemical composition of microalgae can be manipulated readily by changing

the growth conditions, but the effects vary from one species to another. Knowledge ofhow species respond to different environments is of practical use to mariculturists, whomay then grow the algae to optimize the level of specific nutrient(s) needed by thefeeding animal.

Microalgae are important zooplankton food since important algal nutrients (e.g. fattyacids and vitamins) may be transferred to higher trophic levels via the zooplanktonintermediates.

Acknowledgements

We thank Jeannie-Marie Leroi for growing the algal cultures, Kelly Miller, SuzanneNorwood and Christine Farmer (CSIRO Division of Fisheries) for assistance with grosscomposition, sugar, amino acid and vitamin analyses, Stephanie Barrett (CSIRO Divi-sion of Oceanography) for assistance with lipid class analyses. This research wassupported by Grants 86/81, 88/69, 90/63 and 91/59 from the Fisheries Research andDevelopment Corporation, Grant A 1883 1836 from the Australian Research Council anda Rural Credits Development Grant.

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